Chromatin based gene regulation in T lymphocytes

描述(申请人提供)：现在很明显，经典转录控制序列(启动子、增强子、抑制子)直接影响RNA聚合酶活性的能力不能完全解释整个动物染色质中基因表达的组织特异性模式。然而，这些知识对于理解淋巴细胞分化和开发在染色质环境中有效的基因治疗载体是必不可少的。对转基因小鼠转录调控的研究已经发现了一类新的基因调控序列，它们具有明显的基于染色质的活性，不同于经典的转录增强子。一个这样的元素是轨迹控制区(LCR)。我们正在研究小鼠T细胞受体(TCR)-α/Dad1基因座上的LCR。我们目前的长期目标是解释这种LCR活性的分子基础，以及它在调节其复杂基因座中的作用。通过这一研究，我们旨在加深对T细胞分化的基本了解，并为基因治疗载体的开发提供帮助。我们已经确定了该LCR基于染色质的活性的一个关键成分，称为DNA酶超敏感位点(HS)-6。我们假设，hs6等元件参与了最近描述的表观遗传控制机制对其基因座的靶向作用。 在这个项目中，我们利用小鼠的转基因报告基因，目的是确定hs6内与体内LCR活性有关的功能序列和相关因素。我们还旨在鉴定hs6功能序列的作用机制。我们将使用实验室开发的标准和新方法相结合的方法，检测hs6突变对报告基因染色质结构、体内因子占有率、DNA甲基化、组蛋白修饰和核内定位模式的影响。我们的初步数据已经通过体内足迹确定了hs6的两个功能区和三个因子结合位点。LCR中这些位点的突变会损害其基于染色质的活性。因此，这些将是拟议实验的重点。通过提供LCR活性和基于染色质的基因调控的分子基础的清晰图景，这些研究将有助于弥合我们对T细胞特异性基因表达如何实现的了解的差距。这些信息可能有助于改进针对先天性免疫疾病、白血病和注意力缺陷多动障碍的基因治疗策略。

DESCRIPTION (provided by applicant): It is now apparent that the ability of classical transcriptional control sequences (promoters, enhancers, silencers) to directly affect RNA polymerase activity does not fully explain the tissue-specific patterns of gene expression in chromatin of the whole animal. Yet this knowledge is essential to the understanding of lymphocyte differentiation and the development of gene therapy vectors that are effective in a chromatin environment. The study of transcriptional regulation in transgenic mice has identified a novel class of gene regulatory sequences with apparent chromatin-based activities distinct from those of classical transcriptional enhancers. One such element is the Locus Control Region (LCR). We are studying the LCR in the mouse T cell receptor (TCR)-alpha/Dad1 gene locus. Our current long-term goal is to explain the molecular basis for this LCR's activity and its role in the regulation of its complex locus. Through this, we aim to further the basic understanding of T cell differentiation and aid the development of gene therapy vectors. We have identified a key component of this LCR's chromatin-based activity, named DNase hypersensitive site (HS)-6. We hypothesize that elements such as HS6 are involved in targeting recently described epigenetic control mechanisms to its gene locus. In this project, using well characterized reporter transgenes in mice, we aim to determine the functional sequences and associated factors within HS6 contributing to LCR activity in vivo. We also aim to identify the mechanism of action of functional HS6 sequences. Using a combination of standard and novel methods developed in our laboratory, we will examine the effect of HS6 mutations on reporter transgene chromatin structure, in vivo factor occupancy, DNA methylation, histone modification and intra-nuclear localization patterns. Our preliminary data have identified two functional regions and three factor-binding sites in HS6 by in vivo footprinting. Mutation of these sites in the LCR impairs its chromatin based activity. Thus, these will be the focus of the proposed experiments. By providing a clear picture of the molecular bases of LCR activity and chromatin based gene regulation, these investigations will help close the gap in our knowledge of how T cell specific gene expression is achieved. This information may help improve gene therapy strategies against congenital immune disorders, leukemia and A.I.D.S.